The general goal of this proposal is to develop our knowledge of the basic mechanisms of axon extension and neurotransmission as they relate to signaling through Rho-family GTPases. Rho GTPase pathway defects in Drosophila and C. elegans affect both axon pathfinding during the development of the nervous system and neurotransmission in adult animals. These defects dramatically alter locomotory behavior in C. elegans, and in humans Rho pathway defects can result in severe cognitive disorders. Therefore, a better understanding of Rho-family GTPase signaling will further our knowledge of the molecular mechanisms underlying behavior and will facilitate the development of treatments for those with spinal cord injuries or behavioral disorders. The present study is designed to use the model organism C. elegans to examine signaling involving the Rho family GTPase activator UNC-73/Trio in both axon guidance and neurotransmission. My previous work established that the unc-73 locus is complex and encodes two distinct RhoGEF domains in several different protein isoforms. The UNC-73 B isoform contains the Rac GTPase specific RhoGEF-1 domain and functions in axon guidance. The UNC-73 C1, C2 and E isoforms function in neurotransmission and contain the Rho specific RhoGEF-2 domain. The specific aims of this proposal are to identify and characterize molecules that function with UNC-73 in Rho GTPase pathways and to better define how the Rho GTPase pathways function in axon guidance and neurotransmission. Preliminary experiments identified two important UNC-73 B interacting proteins, POD-1 and GEI-18, that are each involved in the regulation of actin polymerization and membrane trafficking events and therefore provide insight into the mechanisms of Rac pathway functions in axon guidance. The first specific aim is to characterize the POD-1 and GEI- 18 proteins and their interaction with UNC-73 using protein/protein binding assays, in vivo co- localization and genetic analysis. The second and third specific aims focus on the role of the UNC-73 isoforms in the process of neurotransmission. Intriguing preliminary data places the UNC-73 RhoGEF- 2 isoforms in a genetic pathway of synapse regulation upstream of the G1s heterotrimeric G protein. My hypothesis is that UNC-73 RhoGEF-2 isoforms function in a neuromodulator signaling pathway to influence neurotransmission. This will be tested by analyzing: 1) specific unc-73 mutants in aldicarb sensitivity assays, 2) neuromodulator production in specific neurons in these mutants and 3) the phenotypes of unc-73 double mutants containing mutations in genes involved in neuropeptide signaling. In addition a genetic screen for suppressors of an unc-73 neurotransmission defective mutant will be performed to identify additional components of the UNC-73 signaling pathway. PUBLIC HEALTH RELEVANCE The goal of this proposal is to develop our knowledge of the mechanisms by which neurons, the cells of the nervous system, are assembled into a complex circuitry in the process of axon guidance and how these cells communicate with each other in the process of neurotransmission. A detailed understanding of these essential processes will eventually allow us to devise better treatments for those with spinal cord injuries or neurological disorders that affect behavior. [unreadable] [unreadable]